Microparticles composed of a polymer are used in various fields due to the ease of controlling their particle size, mechanical strength, particle size distribution, shape and degree of aggregation, examples of which include toner, anti-blocking materials of packing materials, insulating fillers, crystal nucleating agents, chromatographic fillers and abrasives. More recently, microparticles have also been applied to applications such as carriers for immunodiagnostic reagents, spacers of liquid crystal displays, standard particles for calibration of analytical equipment and standard particles for testing of porous films.
The amount of microparticles composed of a polymer used in immunodiagnostic reagent carrier applications in particular is increasing, and the amount used is increasing especially in diagnosis methods using immunochromatographic methods (to be referred to as “immunochromatography”). Although one of the foremost factors behind this increase is the release of large numbers of kits such as home pregnancy kits that are sold as quasi drugs and used by ordinary persons other than health care professionals, these increases have also been driven by a growing demand as means for various types of point-of-care testing (POCT: testing performed in proximity to a patient by a physician or other health care professional that enables results to be obtained rapidly) such as testing for various viruses such as adenovirus, rotavirus or norovirus, hepatitis B, hepatitis C and other types of hepatitis testing, or pathogen testing for pathogens such as E. coli O-157. The number of immunochromatographic tests performed is predicted to increase rapidly in the future in consideration of the growing number of influenza outbreaks in recent years. Microparticles are also being used for immunochromatography in various fields such as biochemical analyses, genetic analyses and other arbitrary analytical reactions in addition to immunodiagnostics.
Immunochromatography is carried out by, for example, selectively reacting an antibody or antigen (ligand) labeled with chromogenic microparticles composed of a metal colloid or colored latex derived from polystyrene with a test substance on a chromatography substrate, and developing while forming a complex. Next, an antigen or antibody (that which specifically binds with the aforementioned ligand) is immobilized in advance on a chromatography substrate at a prescribed detected location, and color is developed by capturing the developed complex. Although various methods have been examined and methods have been established for use as simple testing method, there is a desire to further increase sensitivity and accelerate the diagnostic process based on the need to reduce the burden on health care personnel in the clinical setting when performing POCT.
When diagnosing influenza, there are cases in which a positive result is not obtained by immunochromatography in the early stages of infection despite a positive result being obtained on the following day. It is necessary to further increase the sensitivity of testing in order to solve this problem. In addition, it is becoming increasingly common to simultaneously diagnose both type A antigen and type B antigen with a single immunochromatography kit. In such cases in which multiple specimens are present, although being able to simultaneously test a plurality of test substances with a single test leads to rapid diagnosis, it is also necessary to improve visibility to prevent erroneous diagnoses. Thus, it is preferable that different colors be generated (multicoloration) for each test substance when test substances are detected. Simultaneous diagnosis of multiple test substances using a single kit is also desirable when diagnosis various types of viral infections and when testing food safety, and similar multicoloration is considered to be effective in these applications as well.
The color generated in immunochromatography is derived from the substance used for labeling. In the case of metal colloids, since color is generated due to plasmon effects corresponding to the type of metal thereof, the resulting color is limited to a single color. For example, only red color is generated in the case of using gold colloid as described in Patent Document 1 indicated below. When assuming simultaneous testing of multiple parameters, although effects can be expected to a certain degree by making contrivances to the detected location, this cannot be said to be preferable from the viewpoints of visibility and preventing erroneous diagnoses.
In addition, in the case of metal colloids, a principle referred to as physical adsorption is typically used for the ligand binding method. Typical examples of ligand binding methods include physical adsorption, chemical bonding (covalent bonding), ionic bonding and inclusion. Physical adsorption refers to a binding method that utilizes hydrophobic interaction acting between a base material (such as chromogenic fine particles) and a material to be bound (such as a ligand). In actuality, various mechanisms such as electrostatic action, intermolecular forces and other mechanisms are thought to be acting in addition to hydrophobic interaction. Physical adsorption is advantageous in terms of ease of the procedure and cost since the procedure can be carried out more easily than other binding methods. However, in the case of physical adsorption, there are cases in which problems such as the absence of a fixed binding site and inhibition of adsorption in the presence of a surfactant can occur. In addition, there are also cases in which an adequate amount of ligand cannot be bound.
On the other hand, as disclosed in Patent Document 2 indicated below, in the case of using polystyrene or other latex particles, multicoloration is possible by using a chromophore composed of a disperse dye, oil-soluble dye or pigment. In addition, an arbitrary method such as physical adsorption or chemical bonding can be typically selected for the method used to bind the ligand. Consequently, problems associated with the aforementioned physical adsorption and the like can also be avoided. However, according the examples disclosed in Patent Document 2, the dyeing capacity of the particles is low at about 6% by weight, and the resulting color intensity is weak. Consequently, in the case of using for immunochromatography, it is not possible to obtain distinct coloring effects, thereby resulting in a lack of reliability.
Although Patent Document 3 indicated below discloses microparticles obtained by staining cellulose, since the dyeing capacity relative to the amount of cellulose microparticles is low at about 20% by weight, the resulting stained microparticles are lightly colored. Although these microparticles are used in immunochromatography by imparting an antibody by physical adsorption or chemical bonding as described in Patent Document 4 indicated below, since the amount of antibody bound is insufficient and coloring of the microparticles per se is weak, distinct coloring results are unable to be obtained.